Chromatin Structure In Regulation Of Mammalian Gene Expr

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We study how enhancers activate transcription in the chromatin environment of eukaryotic cells. The human beta-globin genes are activated by an enhancer/locus control region that lies between 6 and 60 Kb distant from the genes themselves. How the strong enhancer activity of the distant LCR is manifest at the promoters of these genes is not well understood, but it encompasses altering chromatin structure. To investigate how the LCR changes chromatin, we are studying a model 5 Kb globin locus on viral episomes in human erythroid K562 cells. We linked the strong HS2 enhancer component of the LCR to the epsilon globin gene, which is actively transcribed from the endogenous locus in these cells. Two types of complexes have been described that carry out chromatin modifications to relieve nucleosomal repression: SWI/SNF complexes that alter nucleosome stability or position, and histone acetyltransferase (HAT) complexes that acetylate histone N-terminal tails. To explore the order of events underlying gene activation by the LCR, we assayed nucleosome mobilization and hyperacetylation at active epsilon globin genes linked to wild type HS2, and compared them to an enhancerless gene, or one linked to an inactivated HS2. We determined that HS2 and the TATA box are required for SWI/SNF mobilization and HAT hyperacetylation at the epsilon globin promoter. BAF 155 is one of the components of SWI/SNF that is involved. We found that SWI/SNF acts first at this promoter, before hyperacetylation occurs. Our additional studies on association of transcription factors and pol II with the enhancer and promoter support the idea that these regulatory elements are in close proximity to each other when transcription is active. We continue to investigate chromatin changes induced throughout our model locus by the HS2 enhancer, and how a chromatin insulator might interrupt this process.